CELLULAR HYPERTROPHY AND CALCIFICATION OF EMBRYONAL CARCINOMA-DERIVEDCHONDROGENIC CELL-LINE ATDC5 IN-VITRO

During the process of endochondral bone formation, proliferating chond rocytes give rise to hypertrophic cells, which then deposit a minerali zed matrix to form calcified cartilage prior to replacement by bone, P reviously, we reported that a clonal cell line, ATDC5, undergoes effic ient chondrogenic differentiation through a cellular condensation stag e, Here we report that the differentiated ATDC5 cells became hypertrop hic at the center of cartilage nodules, when the cells ceased to grow Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline p hosphate (ALPase) activity. After 5 weeks of culture, mineralization o f the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of beta-glycerophos phate, Electron microscopy and electron probe microanalysis revealed t hat calcification was first initiated at matrix vesicles in the territ orial matrix and that it advanced progressively along the collagen fib ers in a manner similar to that which occurs in vivo, The infrared spe ctrum of the mineralized nodules indicated two absorption doublets aro und 1030 cm(-1) and 600 cm(-1), which are characteristic of apatitic m ineral, Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner, Thus, we demonstrated that ATDC5 cells keep track of the multistep dif ferentiation process encompassing the stages from mesenchymal condensa tion to calcification in vitro, ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage d ifferentiation during endochondral bone formation.